Journal
THERIOGENOLOGY
Volume 192, Issue -, Pages 22-27Publisher
ELSEVIER SCIENCE INC
DOI: 10.1016/j.theriogenology.2022.08.021
Keywords
CRISPR-Cas9; Electroporation; Gene editing; Germline transmission; MSTN
Categories
Funding
- National Research Foundation of Korea [2017R1A2B3004972, NRF-2021R1A5A1 033157]
- Research Institute of Veterinary Science
- BK21 Four for Future Veterinary Medicine Leading Education and Research Center
- Seoul National University (SNU) [550-2020005]
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This study assessed the germline transmission of MSTN mutations in F0 cattle and demonstrated successful transmission of the mutations to the next generation. Additionally, the study showed the possibility of further gene editing using electroporation in embryos fertilized with the mutant sperms.
Although the production of several founder animals (F0) for gene editing in livestock has been reported in cattle, very few studies have assessed germline transmission to the next generation due to the long sexual maturation and gestation periods. The present study aimed to assess the germline transmission of MSTN mutations (-12bps deletion) in MSTN mutant F0 male and female cattle. For this purpose, oocytes and semen were collected after the sexual maturation of MSTN cattle, and embryos produced by in vitro fertilization were analyzed. In addition, the embryos were subjected to additional gene (PRNP) editing using electroporation. Embryos produced by in vitro fertilization with MSTN male and female cattle were transferred to a surrogate, and one calf was successfully born. MSTN heterozygous mutation was shown by sequencing of the F1 calf, which had no health issues. As a further experiment, using electroporation, additional gene-edited embryos fertilized with the MSTN male sperm showed a high mutation rate of PRNP (86.2 +/- 3.4%). These data demonstrate that the cattle produced through gene editing matured without health issues and had transmitted MSTN mutation from the germ cells. Also, additional mutation of embryos fertilized with the MSTN male sperm could enable further mutagenesis using electroporation.(c) 2022 Elsevier Inc. All rights reserved.
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